The regulatory agencies responsible for protecting patients and the public from potential heath hazards caused by dental materials have a crisis caused by the absence of suitable screening assays to measure how the dental materials interact with oral cells. For over a quarter of a century, the cellular cytotoxicity in vitro testing standard has been set by the American National Standards Institute/American Dental Association (ANSI/ADA) Document No. 41a (1982). Although a new standard is pending, it will advocate the same in vitro test that has been used since the 1950's. The standard allows for the contact testing of solid dental materials for cytotoxicity with L929 mouse fibroblasts. The same standard is applied worldwide for all new biomaterials, devices and therapies in guidelines 7405 and 10993, formulated by the International Organization for Standardization (ISO). The ISO recommended the development of new investigative approaches to test cytoxicity that have a clear translational relevance to the clinical situation. But none have been developed so far. The objective of this particular application is to create improved cytotoxicity screening assays that are more clinically-relevant to dentistry, than the ISO-recommended assays currently employed. The central hypothesis is that new assay's which are more relevant to dentistry will improve the clinical relevance of the cytotoxicity screening of dental materials. The rationale for the proposed research is to create new cytotoxicity screening assays for biomaterials that use dental pulp and periodontal cells, and to incorporate these screening protocols into the ADA and ISO guidelines. Guided by strong preliminary data, this hypothesis will be tested by pursuing two Specific Aims: 1) test the hypothesis that the current ISO guidelines for cytotoxicity screening cannot be translated as a reliable measure of toxicity when a dental material is used clinically;and 2) to test the hypothesis that there is a difference in the cytotoxicity of different classes of dental materials, and a difference in the sensitivity of different types of cells to dental materials. The proposed research is relevant to that part of NIH's mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human disability. Our approach is innovative, because we propose to update the ANSI/ADA/ISO guidelines to use human oral cell lines, instead of L929 mouse fibroblast cells. There seems to be no justification to continue using old mouse cell lines which are non-specific to dentistry to test the safety of dental materials. The use of cytotoxicity screening assays incorporating dental cells is expected to increase the translational relevance of the assays to dentistry, and thereby increase the clinical accuracy of cytotoxicity measurements of dental materials. The proposed research is significant, because dental materials are placed in the mouths of hundreds of millions of American patients each year: New more clinically relevant cytotoxicity screening assays are needed to protect them from avoidable health risks. Public Health Relevance: The adoption of these new guidelines will improve the translation of cytotoxicity screening assays to clinical dentistry, and open a new pathway to develop improved biomaterials. This project will update the cytotoxicity screening standards for the 21st century and benefit the general public by promoting the development of safer dental biomaterials, thereby protecting them from dental materials health risks.